Assessment of a one-week ketogenic diet on brain glycolytic metabolism and on the status epilepticus stage of a lithium–pilocarpine rat model

The ketogenic diet (KD) has been shown to be effective in refractory epilepsy after long-term administration. However, its interference with short-term brain metabolism and its involvement in the early process leading to epilepsy remain poorly understood. This study aimed to assess the effect of a short-term ketogenic diet on cerebral glucose metabolic changes, before and after status epilepticus (SE) in rats, by using [18F]-FDG PET. Thirty-nine rats were subjected to a one-week KD (KD-rats, n = 24) or to a standard diet (SD-rats, n = 15) before the induction of a status epilepticus (SE) by lithium-pilocarpine administrations. Brain [18F]-FDG PET scans were performed before and 4 h after this induction. Morphological MRIs were acquired and used to spatially normalize the PET images which were then analyzed voxel-wisely using a statistical parametric-based method. Twenty-six rats were analyzed (KD-rats, n = 15; SD-rats, n = 11). The 7 days of the KD were associated with significant increases in the plasma β-hydroxybutyrate level, but with an unchanged glycemia. The PET images, recorded after the KD and before SE induction, showed an increased metabolism within sites involved in the appetitive behaviors: hypothalamic areas and periaqueductal gray, whereas no area of decreased metabolism was observed. At the 4th hour following the SE induction, large metabolism increases were observed in the KD- and SD-rats in areas known to be involved in the epileptogenesis process late—i.e., the hippocampus, parahippocampic, thalamic and hypothalamic areas, the periaqueductal gray, and the limbic structures (and in the motor cortex for the KD-rats only). However, no statistically significant difference was observed when comparing SD and KD groups at the 4th hour following the SE induction. A one-week ketogenic diet does not prevent the status epilepticus (SE) and associated metabolic brain abnormalities in the lithium-pilocarpine rat model. Further explorations are needed to determine whether a significant prevention could be achieved by more prolonged ketogenic diets and by testing this diet in less severe experimental models, and moreover, to analyze the diet effects on the later and chronic stages leading to epileptogenesis.

Therefore, KD remains an attractive option especially, in children and adolescents with refractory epilepsy for whom surgery was ruled out [9][10][11] .Recently, international experts recommend the initiation of the KD within the first week after the diagnosis during the acute phase, with the need to be continued for up to 3 months (postacute phase) if proven to be effective 12 .
In animal studies, the effect of ketogenic diet on epilepsy treatment has been already studied, often involving its administration before inducing a status epilepticus [13][14][15][16][17] .Neurons and astrocytes exhibit the capacity to absorb and catabolize β-hydroxybutyrate and acetoacetate, using ketone bodies in mitochondria for energy production and amino acid synthesis [18][19][20] .
In vivo imaging techniques, such as [ 18 F]-fluorodeoxyglucose positron emission tomography ([ 18 F]-FDG PET) and magnetic resonance imaging (MRI) can be used as two complementary noninvasive imaging techniques to evaluate brain metabolic changes, particularly the glucose metabolism, at an early stage of epilepsy 4,21 .Their combination provides functional information from molecular PET imaging and morphological information from the MRI.Their coupling with quantitative analyses brings new diagnostic solutions by giving complementary elements on the detection of small brain abnormalities and on the characterization of the seizure location 4,[21][22][23] .In a previous study from our team, the changes in the brain glycolytic metabolism have been characterized and quantified using [ 18 F]-fluorodeoxyglucose positron emission tomography (PET) imaging in a lithium-pilocarpine epilepsy model, displaying an early hypermetabolism on site known to be involved in the epileptogenesis process (piriform and entorhinal cortex, hippocampus) 21 .
One of the most recognized method for semi-quantitative analysis of neuroimaging data is Statistical Parametric Mapping (SPM), widely used in preclinical studies 21,24 .SPM also tends to improve the visual assessment in various clinical studies [25][26][27][28] .It improves reproducibility and is a time saver compared to visual analyses.
By using [ 18 F]-FDG PET and a dedicated voxel-based quantification pipeline, the present study aimed to assess the effect of a short-term ketogenic diet (7 days) on cerebral metabolic changes, before and after the status epilepticus (SE) and especially within the brain areas known to be involved in the later stages of epiletogenesis development.
The paired comparisons of the PET images before (D-8) and after (D-1) the KD showed areas of significant enhanced glycolytic brain metabolism at D-1, mainly within the hypothalamus (increased metabolism volume = 21.54mm3T-voxel max at 5.21) and the periaqueductal grey matter (9.88 mm3, T-voxel max at 5.76) In contrast, no hypometabolic areas at D-1 were observed.The details are reported in Fig. 1 and Table 2.

SE after the ketogenic or standard diets
On the day of the SE induction, the level of plasma β-hydroxybutyrate was higher for the KD-rats than for the SD-rats (1.67 ± 0.37 vs. 1.08 ± 0.3 mmol/L, p < 0.05), but there was no significant difference between these 2 groups, neither for the body weight (321 ± 25 vs. 358 ± 31 g, NS), nor for the glycemia (6.59 ± 1.11 vs. 6.72 ± 0.25 mmol/L, NS) (Table 3).
All pilocarpine-treated rats entered convulsive SE of which 4 SD rats died during or after 4 h of convulsive SE.The characteristics of the lithium pilocarpine-induced SE state were not influenced by the ketogenic diet.In each group, the event characteristics were like those previously documented in this model [29][30][31] .The rats of all groups exhibited the same behavioral features after the lithium-pilocarpine administration.Within 5 min after the pilocarpine injection, the rats developed diarrhea, piloerection, and other signs of cholinergic stimulation.During the following 10-20 min, the rats exhibited head bobbing, scratching, chewing, and exploratory behavior.Recurrent seizures started after pilocarpine administration with associated episodes of head and bilateral forelimb myoclonus with the animals rearing up on their hind legs and falling.They progressed to SE around 30-40 min after the pilocarpine injection.
The recurrent seizure thresholds seemed to appear faster in KD rats compared to SD rats (12-30 min after the pilocarpine injection vs. 18 to 32 min, in the KD and SD rats respectively, p < 0.05).The SE severity, however, was similar between the two groups.
The results of the paired comparisons of PET images recorded before the SE (D-8) and at the 4th hour following the SE induction (H+4), are detailed in Fig. 2 for both groups and in Tables 4, 5 and Tables 6, 7 for the SD-and KD-rats' groups, respectively.Both groups exhibited equivalent increases of the brain metabolism and within the same brain areas (Tables 4 and 6)., i.e., hippocampus (KD-rats: 71.83 mm ).An increase of the brain metabolism was observed on the motor cortex for KD-rats (32.57mm 3 , 8.23).Hypometabolic areas were also observed in some areas as in the corpus callosum or in the cingular cortex (Table 5 and 7).No significant differences were observed for the unpaired comparisons between the [ 18 F]-FDG PET images from SD-and KD-rats at the 4th hour following SE induction.

Discussion
After status epilepticus (SE), a dynamic process start, leading to an abnormal brain network reorganization 32 , metabolic changes and neurodegeneration, contributing to the establishment of the chronic disease.On the rat lithium-pilocarpine model, a brain glycolytic hypermetabolism occurring 1 to 4 h after SE, has already been documented in the areas known to be involved in the subsequent epileptogenesis process (mostly in the regions related to SE propagation as piriform cortex, amygdala, CA1 of the hippocampus, and the hilus of dentate gyrus (DG)) 21,29,30 .
Therefore, by using [ 18 F]-FDG-PET and a dedicated voxel-based quantification pipeline, we attempted to investigate the impact of a short-term ketogenic diet (7 days) on the brain glycolytic metabolism, in a well-known Table 2. Anatomical localization, volume and maximal T-scores of the areas of increased glycolytic metabolism observed with the semi-quantitative analysis in the 15 KD-rats after 7 days of KD (i.e., with a paired comparison between D-8 and D-1 PET images).Only the areas with a T-voxel > 3.79 and a cluster size > 38 voxels are reported.

Anatomical location
Volume (mm and validated model of TLE, the rat lithium-pilocarpine model, with a special focus on the metabolism of the brain areas known to be involved in the subsequent epileptogenesis process.
To reduce the variation in glucose metabolism caused by a KD, we tested the effects of this diet on the brain metabolism in healthy rats.The diet was well tolerated, with the animals considerably gaining weight within one week.The blood level of the ketone bodies was also significantly increased, confirming previous works reporting a drastic carbohydrate reduction during 3 to 5 days, can induce a ketosis 15,33 .However, we noted that glycemia remained unchanged despite the 7-day low-carbohydrate diet in accordance with results from previously published studies [34][35][36] .
We used a voxel-based method for the PET images analysis (SPM), which provides an objective semiquantification of the brain metabolic changes.We specifically designed a dedicated SPM pipeline for this study.The spatial normalization step using anatomical MRI images guarantees the best localization and ensures a total independence of the normalization and statistical analyses 37 .It is not the case when the voxel information from the PET scans is used in the two steps.Actually, it might lead to a loss of spatial accuracy and to a dispersion of the voxel values and then, to a loss of power of the statistical analysis 38,39 .This statement is particularly relevant in our study for H+4 acquired images.In fact, the important hypermetabolism during SE is responsible for large differences between the to-be-normalized images and the PET template.The intensity normalization is also a crucial step in [ 18 F]-FDG semi-quantitative studies.The concentration reaching the brain is subjectdependent because of different physiological factors, such as, among others, the blood glucose level, medications, and age 40 .The proportional scaling is widely used but was not adapted for our study.In fact, a significantly higher global intensity at H+4 (in comparison with D-8) can lead to an attenuation of the hypermetabolism areas and create an artificial hypometabolism in normal areas on H+4 normalized images.In previous studies on the lithium-pilocarpine rodent model 21,41,42 , the pons was recommended as the reference region for the intensity normalization, but we did not use it in our study in order to take in consideration the metabolism variations within the pons due to the ketogenic diet.Opposed to the anatomical reference region methods, the histogram-based method used in this paper is data-driven and efficient to detect abnormal patterns without requiring previous knowledge about the analyzed model or disease 43 .This method has already been used in other preclinical studies 44 and also proved its efficiency in a clinical context 40 .Indeed, although they are present, the artificial hypometabolic areas at H+4 (vs D-8) are limited and considered as false positive because no metabolism decrease during SE is reported in the literature.
This pipeline was firstly applied to the [ 18 F]-FDG PET images recorded in healthy rats before (D-8) and after a 7-days (D-1) ketogenic diet (KD) before the induction of the epilepsy.Paired analyses gave clear evidence of a KD-related hypermetabolism in periaqueductal gray (PAG) and hypothalamic areas.In the brain, these structures are activated through the food intake and reward processes and have extensive connections mediating the appetitive and the consummatory behaviors 45 .The periaqueductal gray matter has long been known as a region modulating fear and anxiety-related reactions [46][47][48][49] .More recently, its role in regulating and driving appetitive behaviors and its connections to the central feeding network of the brain, such as the hypothalamus, have been explored 48,50 .Likewise, previous study has also shown that the inactivation of PAG in rats alters the hypothalamic signaling and the food consumption 45 .Our data provide new evidence that hypothalamic areas and PAG may play a major role in appetitive behaviors and that 7 days of ketogenic diet can induce brain metabolic changes in normal rats.In contrast, we observed no decrease in brain metabolism after a 7-day KD, showing a physiological consumption of glucose in other areas of the brain.This is in accordance with the observation of an unchanged plasma glucose in our KD rats.It must be kept in mind that complex metabolic adaptations may influence glucose regulation during short KD periods.More precisely, it is likely that certain enzymes of glucose metabolism have not yet been significantly downregulated after a 7-day period (i.e., metabolic adjustments could www.nature.com/scientificreports/take a longer time to stabilize) 51 .In addition, it has been postulated that the control of metabolic enzymes and transporters could vary between different brain areas 52 .Finally, as suggested by Stincone et al., the acute use of ketone bodies during KD could lead to a paradoxical increase in the glucose release within blood, in order to maintain important functions such as the cytoplasmic antioxidant defense 51,52 .
After the SE induction, the SPM analysis showed a comparable increased metabolism within the primary sites that are subsequently implicated in the epileptogenesis process (hippocampic and parahippocampic areas, entorhinal and piriform cortices and thalamic and hypothalamic areas) 15,21,30 in the KD group and in the SD Table 4. Anatomical localization, volume and maximal T-scores of the increased glycolytic metabolism areas observed with the semi-quantitative analysis in the 15 KD-rats during SE (i.e., with a paired comparison between the PET images recorded before SE (D-8) and at the 4th hour from SE induction (H+4)).Only the areas with a T-voxel > 3.79 and a cluster size > 4 mm 3 are reported.

Anatomical location
Volume (mm group at H+4 compared to D-8.We observed slightly more pronounced hypermetabolic volumes in the KD group compared to the SD group.Several hypermetabolism areas, were also detected in the KD but not in the SD group, such as motor cortex (Tables 4 and 5).However, the inter-group comparisons do not show a significant difference in brain [ 18 F]-FDG uptake, at H+4 of the SE, between the KD and SD rats using SPM.
The inability of the KD to prevent lithium-pilocarpine SE was already documented.A prior investigation by Linard et al. explored the effects of the ketogenic diet in the lithium-pilocarpine rat model, using an electroencephalogram and histological analysis.Their study demonstrated a significant decrease in hippocampal neuronal loss, specifically in CA1 and CA3 regions, in the KD-rats compared to SD-rats.However, no significant difference were observed in the characteristics of clinical events during the acute phase of lithium-pilocarpine SE between the two groups 15 .

Limitations and perspectives
Although the lithium-pilocarpine model reproduces the main clinical features of human TLE, extrapolation to the human epileptogenesis process remains difficult.In addition, it is likely that the KD effects could be different between rats and humans.
No histopathologic analyses were performed in the present study, which constitutes an additional limit.However, a number of previous studies published on this model, including a study from our team, have shown that most damaged neurons were located in the structures showing a significant increase in the metabolic activity Table 6.Anatomical localization, volume and maximal T-scores of the areas of increased glycolytic metabolism observed with the semi-quantitative analysis in the 11 SD-rats after SE (i.e., with a paired comparison between the PET images recorded before SE (D-8) and at the 4th hour after SE induction (H+4)).Only the areas with a T-voxel > 3.79 and a cluster size > 4 mm 3 are reported.

Anatomical location
Volume (mm www.nature.com/scientificreports/during the status epilepticus 30,[53][54][55][56] .The neuron damages and their potential prevention by a diet would be closely tied to the capacity of neurons to manage the extreme metabolic demands (3 to 6-folds increases) from certain brain regions 57,58 .
In these conditions and despite the preference for ketones, these brain areas should maintain a high glycolytic activity to meet the energy needs of the lithium-pilocarpine model.
As already discussed above, a 7-day KD may not be sufficiently long to complete the transition of the brain into a state of complete ketosis, using ketone bodies as the exclusive energy substrate.Achieving all the requisite metabolic alterations in the brain, might require a more prolonged period of ketogenic intervention. 59,60.
The acute SE produced by the lithium-pilocarpine model is particularly aggressive, and it could be interesting to test KD on model gradually developing clinical and electroencephalographic seizure manifestations, such as the pentylenetetrazol (PTZ) perfusion rat model.
Another limitation is that the glycolytic metabolism assessed with [ 18 F]-FDG-PET would not precisely correspond to the severity of neuronal loss 61 .The area of abnormal metabolism identified by interictal [ 18 F]-FDG-PET often extends well beyond the epileptogenic zone, covering a large portion of the temporal lobe and surrounding regions 55,61 .Consequently, it may be worthy to explore alternative radiotracers such as [ 18 F] flumazenil for a more precise delineation of the epileptogenic areas 62,63 .
A last limitation is that our study did assess the early stage of SE but did not extend to the later and chronic stages of the epileptogenesis process.A continued administration of throughout KD the chronic phase of epilepsy, would have provided a more comprehensive understanding of the KD impact on the epileptogenic process.

Conclusion
A one-week ketogenic diet does not prevent the status epilepticus (SE) and metabolic brain abnormalities in the lithium-pilocarpine rat model.Further explorations are needed to determine whether a significant prevention could be achieved by more prolonged ketogenic diets and by testing this diet in less severe experimental models, and moreover, to analyze the diet effects on the later and chronic stages leading to epileptogenesis.

Animals and study design
All experiments involving animals complied with the ARRIVE guidelines and were approved by the Lorraine Ethics Committee N°66 according to Guidelines of Animal Care and Use (APAFIS no.16424-2018080814271998). Sprague-Dawley male rats (175-200 g) were purchased from Janvier Laboratories (Le Genest-Saint-Isle.France) and housed in ventilated cages including filter tops, under controlled environmental conditions (22 ± 2 °C, 55 ± 20% humidity) and 12-h light-dark cycle.
The imaging timeline is illustrated in Fig. 3.A brain [ 18 F]-FDG PET was recorded in 24 adult male rats (D-8) and then, after 7 days of a KD (D-1, Fig. 3) (3% carbohydrate, 73% fat, 15% protein, 0% fiber, 9% vitamins, and minerals; ketocal ® SDS.France) 31,64,65 .A status epilepticus (SE) was thereafter induced in these KD-rats, with a third [ 18 F]-FDG PET being recorded 4 h after the SE induction (H+4).A total of 24 KD rats were initially included in the study, but 4 rats had to be withdrawn due to technical issues encountered during the PET experiments.Additionally, 5 other rats were excluded from the analysis due to either a lack of MRI scans or the poor quality of PET images.
The same protocols of SE induction and H+4 [ 18 F]-FDG PET recording were applied, for comparisons, in a control group of 15 rats submitted to a standard diet (SD-rats; 44% carbohydrate, 6% fat, 19% protein, 18% fiber, 13% vitamins, and minerals; Envigo.Gannat.France).In these SD-rats, a baseline [ 18 F]-FDG PET scan was also performed, 7 days before SE induction (D-8).Moreover, for both groups, an MRI was realized before D-8.Food and water were given ad libitum in both groups and each rat was weighed daily at a fixed time with a dedicated small-animal scale (Mettler Toledo.DeltaRange-PR5002.France).Before each PET, two drops of blood were taken from the tip of the tail for the measurement of blood glucose and β-hydroxybutyrate levels by using Medisense Optium Xceed reader (Abbott France Division Medisense.Rungis.France) 64 .

Figure 1 .
Figure 1.Study design.D-8 and D-1 are defined as eight and one day before status epilepticus (SE) respectively, H+4 is defined as four hours after SE.

Figure 2 .
Figure 2. Representation of the spatial normalization pipeline.The PET images are normalized using nonlinear transformations estimated between the SIGMA MRI (used to create the Atlas) and the individual MRI of each rat.PET images were previously registered to their individual MRI.

Figure 3 .
Figure 3. Anatomical localization of the areas of increased metabolic activity between Day (-8) and Day (-1) and for KD subjects (n = 11).The SPM-T maps were obtained using a paired test (p < 0.001, uncorrected, k > 50 voxels) then projected onto two-dimensional slices of T1-weighted MRI.The colorbar represents the T-values.

Figure 4 .
Figure 4. Areas of increased [ 18 F]-FDG uptake observed at the 4th hour after induction of SE (i.e., with a paired comparison between the PET images recorded before SE and at the 4 th hours after SE induction) in the 11 SD-rats (left panel) and 15 KD-rats (right panel).SPM-T maps are displayed with a Z score-based color scale, projected on T1-weighted MRI slices, and with the following parameters: p < 0.001 (no correction for multiple comparisons), and k > 38 voxels.The colorbar represents the T-values.

Table 1 .
3, T-voxel max at 15.52 Effect of the diet treatment on the physiological parameters.

Table 3 .
Comparison of physiological parameters between the SD-rats and the KD-rats before the induction of the status epilepticus.

Table 5 .
Anatomical localization, volume and maximal T-scores of the decreased glycolytic metabolism areas observed with the semi-quantitative analysis in the 15 KD-rats during SE (i.e., with a paired comparison between the PET images recorded before SE (D-8) and at the 4 th hour from SE induction (H+4)).Only the areas with a T-voxel > 3.79 and a cluster size > 4 mm 3 are reported.

Table 7 .
Anatomical localization, volume and maximal T-scores of the decreased glycolytic metabolism areas observed with the semi-quantitative analysis in the 11 SD-rats during SE (i.e., with a paired comparison between the PET images recorded before SE (D-8) and at the 4th hour from SE induction (H+4)).Only the areas with a T-voxel > 3.79 and a cluster size > 4 mm 3 are reported.